Journal of Molecular Catalysis B-enzymatic最新文献

Novel magnetic cellulose nanocrystals (MCNCs) prepared via electrostatic self-assembly approach were used as magnetic carriers for efficient immobilization of papain and facilitated recovery of this immobilized enzyme. Zeta potential measurements, Fourier transform infrared spectroscopy and Scanning electron microscope were applied to evaluate the forming mechanism and surface structure of MCNCs. Cellulose nanocrystals (CNCs) were successfully combined with cationic polyethyleneimine (PEI) modified Fe₃O₄ nanoparticles (NPs), and the electrostatic interaction between them was a key driving force. The prepared MCNCs were successfully used for the immobilization and separation of papain from the reaction system. When enzyme concentration and pH value of enzyme solution were 0.4 mg mL⁻¹ and 6, respectively, the resultant immobilized enzyme exhibited the highest enzymatic activity about 227 μg min⁻¹ g⁻¹. Better pH and thermo stabilities than those of the free papain were also achieved after immobilizing the enzyme on MCNCs. Furthermore, the immobilized papain manifested enhanced tolerability to three different solvents, namely n-butyl alcohol, n-hexane and [Cnpy][NTf₂], respectively. The prepared MCNCs as the efficient carrier materials have a strong application potential for enzyme immobilization.

A putrescine N-hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N-hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine > cadaverine > hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4 kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His₁₀-tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent K_m and k_cat of 361.6 ± 0.1 μM and 0.266 ± 0.011 s⁻¹, respectively, whereas the hydroxylation assay showed GorA with an apparent K_m and k_cat of 737.1 ± 0.1 μM and 0.210 ± 0.001 s⁻¹. These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N-hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis.

The peroxygenase from Agrocybe aegerita (AaeUPO) has been evaluated for stereoselective oxyfunctionalization chemistry under non-aqueous reaction conditions.

The stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was performed in neat substrate as reaction medium together with the immobilized biocatalyst and ^tertBuOOH as oxidant.

Stability and activity issues still have to be addressed. Nevertheless, gram-scale production of enantiopure (R)-1-phenylethanol was achieved with respectable 90,000 turnovers of the biocatalyst.

Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of β-d glucose utilizing molecular oxygen as an electron acceptor to produce d-glucono-1,5-lactoneand hydrogen peroxide, which has applications in food, biotechnology and medical industries. It was known that dimer form was considered to be active and monomer form has inactive conformation. However, there are no evidences at the molecular levels for Glucose oxidase (GOx) inactivation through dissociation. Here, using molecular dynamic simulation, it has been investigated for the first time that why dimer form of the enzyme is active. We have performed a series of molecular dynamics simulations at different forms of GOx (monomer and dimer with and without FAD cofactor). The analysis of tertiary structure showed that monomer is more unstable and has more deviation from the crystal structure. In contrast, dimer has a stable conformation during simulation. These results are in good agreement with experimental data about enzyme inactivation by dissociation. It was also found that when FAD is removed from monomer, it became more unstable in comparison with monomer containing cofactor. This shows essential role of FAD in both activity and stability of the enzyme. According to the MD simulation, enzyme inactivation is associated with changing in secondary structure at the interface. Interestingly, it was found that some secondary structures are destructed while some structures are formed in monomer upon dissociation. The analysis of active site structure during simulation revealed that both dissociation and release of the FAD influence on inactivation of GOx. This study provided novel insight to understand the mechanism of enzyme inactivation upon dissociation, which would be useful for rational enzyme design.

Biotransformation of lignocellulose by microbial fermentation is usually preceded by thermo-chemical pretreatments followed by enzymatic hydrolysis of cellulose. Derivatives formed during the pretreatment of the lignocellulosic biomass inhibit enzymatic hydrolysis as well as microbial fermentation. Pretreated lignocellulose hydrolysate contains many derivatives of either furanic or phenolic inhibitory derivatives. In the present study, laccase was used to detoxify three different types of lignocellulosic derivatives that are highly toxic to microbial fermentation due to their low hydrophilic nature, namely furfural, acetosyringone, and coniferyl aldehyde. A minimal inhibitory concentration (MIC) test was carried out with Saccharomyces cerevisiae. The MIC of furfural, acetosyringone, and coniferyl aldehyde was 12 mM, 24 mM, and 1.5 mM, respectively. Laccase was immobilized on to cellulose nanofiber produced by Gluconacetobacter xylinus. Immobilized laccase showed a better pH and thermal stability than free laccase. Reuse of immobilized laccase retains 85% of its enzyme activity after 16 recycles. Immobilized laccase completely degraded the three lignocellulose inhibitory derivatives after 36 h of incubation at 40 °C. Finally, the degradation was confirmed by ultraviolet visible spectroscopy (UV–VIS spectrum), high performance liquid chromatography and liquid chromatography mass spectrometry. Interestingly, it was found that the effect of enzymatic degradation depends on the structural variation of the lignocellulosic derivatives as laccase alone detoxified the furfural and coniferyl aldehyde, whereas a redox mediator HOBt was needed for the detoxification of ketone based lignin derivative acetosyringone.

Besides merely destroying H₂O₂, an important use of the catalase reaction, H₂O₂ → 1/2 O₂ + H₂O, is to supply O₂ to oxygenation reactions. Due to convenient spatiotemporal control over O₂ release, oxygenation from H₂O₂ is useful in particular for enzymatic reactions confined to solid supports. Because commercial catalases are difficult to immobilize, we have developed a one-step procedure of purification and immobilization of Bordetella pertussis catalase, recombinantly produced in Escherichia coli. Fusion of the catalase to a positively charged binding module enabled effective immobilization of the chimeric enzyme on anionic support (Relisorb SP 400), giving a controllable activity loading of between 5000 and 100,000 units/g support. Use of the immobilized catalase in combination with H₂O₂ feeding provided O₂ to the reaction of glucose oxidase in solution for a range of volumetric conversion rates (0.2–1.5 mM/min). Using optical sensing to measure the O₂ concentration in the liquid but also in the solid phase, we showed that internal superoxygenation of the support was made possible under these conditions, resulting in an inverted (that is, negative) O₂ concentration gradient between the bulk and the particle and allowing the internal O₂ concentration to exceed by up to 4-fold the limit of atmospheric-pressure air saturation in solution. By tailored immobilization of B. pertussis catalase, therefore, an efficient biocatalytic system for hydrogen peroxide conversion in porous solid support was developed. This could find application for bubble-free oxygenation of O₂-dependent enzymes co-immobilized with the catalase whereby enhanced internal availability of O₂ would contribute to biocatalytic reaction intensification.

Eight new isolated fungi of the genus Penicillium were evaluated for β- fructofuranosidase (FFase) production. From these, Penicillium citreonigrum was selected for FFase and fructooligosaccharides (FOS) production. The influence of temperature, yeast extract concentration, pH and fermentation time on the FFase activity when using the whole microorganism was evaluated by 2⁴ and 2³ designs. The pH was set at 6.5 and no yeast extract was used in the optimization experiments since both shown low significant effects on FFase activity. After optimization, temperature and fermentation time, were set to 25.5 °C and 67.8 h. Under these conditions, the model predicted a FFase production of 301.84 U/mL. The scaled-up process in a 2 L bioreactor enhanced the enzyme productivity up to 1.5 times (6.11 U/mL h). A concentration of 58.7 g/L of FOS was obtained, where kestose was the main product. Assays performed for enzyme characterization showed that 50 °C and a pH 5.0 are the optimal conditions for FFase activity. FFase showed to be stable at temperatures between 25 and 30 °C and pH 4.0–10.0 and its activity increased in the presence of ions, especially Cu⁴⁺. Results obtained in this primary report are a clear indication on the interest of using P. citreonigrum as a source of FFase for further FOS production.

Ethyl (R)-4-chloro-3-hydroxybutanoate ester [(R)-CHBE] is an important chiral intermediate for the synthesis of chiral drugs. In this study, a novel short-chain, NADH-dependent dehydrogenase (LCRIII) from Lactobacillus curieae S1L19 was discovered to exhibit high activity and enantioselectivity in the production of (R)-CHBE by reduction of ethyl 4-chloroacetoacetate (COBE). LCRIII was heterologously overexpressed in Escherichia coli and the protein was purified to homogeneity. Characterization of LCRIII showed broad substrate specificity towards a variety of ketones. In addition, an efficient cofactor regeneration system was constructed by co-expressing LCRIII and glucose dehydrogenase (GDH) in E. coli cells. Up to 1.5 M (246.8 g/L) COBE could be completely reduced to (R)-CHBE with excellent enantiomeric excess ( > 99% ee) in a monophasic aqueous system. Moreover, the process could be performed even without external addition of cofactors. These results demonstrate the great potential of this process in industrial applications.